The paramyxovirus family of negative stranded enveloped RNA viruses contains highly contagious, clinically important pathogens such as measles virus (MV), respiratory syncytial virus, and human parainfluenza viruses (hPIV), and the recently emerged highly pathogenic Nipah and Hendra viruses (Wolfson et al., (2007) Lancet 369, 191-200; CDC. (2005) MMWR 54(8), 200-203)
MV remains a principal cause of worldwide morbidity and mortality, being responsible for approximately 300,000 to 400,000 deaths annually, despite the existence of a live-attenuated vaccine. Globally, measles is the leading cause of childhood death from a vaccine-preventable disease and remains among the ten most lethal human pathogens. Transmitted via the respiratory route, the virus is highly communicable and one of the most infectious pathogens identified (Griffin, D. E. (2001) Measles Virus, 4 Ed., Lippincott, Philadelphia, Pa.; Hethcote, H. W. (2000) SIAM Review 42(4), 599-653; van den Hof et al., (2002) J. Infect Dis. 186(10), 1483-1486). Prolonged immunosuppression following acute cases frequently predisposes patients to bacterial otitis media and bronchopneumonia. Complications include acute encephalitis in approximately 0.1% of cases, and subacute sclerosing panencephalitis (SSPE), a lethal late sequelae that occurs years after the primary infection (Griffin, D. E. (2001) Measles Virus, 4 Ed., Lippincott, Philadelphia, Pa.; Hilleman, M. R. (2001) Vaccine 20(5-6), 651-665).
Despite ongoing efforts to ultimately eradicate the virus (Moss & Griffin. (2006) Nat. Rev. Microbiol. 4(12), 900-908; Ota et al., (2005) J. Neurovirol. 11(5), 447-454), several factors contribute to the global persistence of MV and its resulting morbidity and mortality. It is estimated that a herd immunity of greater than 95% is required for complete suppression of the virus (Hethcote, H. W. (2000) SIAM Review 42(4), 599-653; Moss & Griffin. (2006) Nat. Rev. Microbiol. 4(12), 900-908; Ota et al., (2005) J. Neurovirol. 11(5), 447-454),). Maintaining fully protective herd immunity requires repeated vaccination since administration of a single dose at 12 months of age is not sufficient to meet this goal (Meissner et al., (2004) Pediatrics 114(4), 1065-1069; Watson et al., (1998) MMWR Recomm. Rep. 47(RR-8), 1-57). This constitutes a particular challenge in the developing world. In the US, a second dose of the vaccine is recommended for all school age children Pediatrics, A. A. O. (1989) Pediatrics 84, 2220-1113). Although high coverage rates are more readily achievable in developed countries, herd immunity has dropped significantly below the 95% level in several countries due to parental concerns about vaccine safety, resulting in lowered vaccination compliance. In recent years, coverage in certain areas of Europe has declined to less than 80%, resulting in significant measles outbreaks with a corresponding increase in hospitalizations and measles-associated deaths (van den Hof et al., (2002) J Infect Dis 186(10), 1483-1486; Jansen et al., (2003) Science 301(5634), 804; McBrien et al., (2003) Pediatr. Infect. Dis. J 22(7), 580-584). Lastly, immunity against the attenuated vaccine strain is less durable than that acquired naturally (Putz et al., (2003) Int J Parasitol 33(5-6), 525-545). In a fully vaccinated population, natural boosting by circulating wild-type virus is absent and half-lives of protective antibodies have been estimated at 25 years or less (Mossong et al., (1999) Am J Epidemiol 150(11), 1238-1249; Mossong et al., (2000) Vaccine 19(4-5), 523-529). In this environment of waning immunity, re-introduction of circulating virus in the population may be facilitated by individuals with weak immunity who may be protected against disease but not against infection (de Swart et al (2000) Lancet 355(9199), 201-202; Whittle et al., (1999) Lancet 353(9147), 98-102), thus creating a basis for spontaneous outbreaks.
The only technology presently available to prevent measles virus infection is vaccination. Immunity takes weeks to develop, and vaccination is contra-indicated in immune compromised individuals. The current vaccines cannot be administered to infants due to interfering of maternal antibodies. Therapeutics for case management of measles and the rapid control of measles outbreaks are not available. For Nipah virus, no therapeutic or prophylactic strategies are in place. Taken together, these factors make highly desirable the development of cost-effective therapeutics against MV that augment the existing vaccination program by helping to control local outbreaks and manage cases of severe measles. Small molecule entry inhibitors could be made readily available to confer immediate protection, and could be safely administered to immune compromised patients to control acute MV or Nipah virus infection. These molecules could also be beneficial in treatment of complications of measles virus infection, such as the lethal sequelae subacute sclerosing panencephalitis.
MV infection is initiated by pH-independent fusion of the viral envelope with the target cell plasma membrane (Griffin, D. E. (2001) Measles Virus, 4 Ed., Lippincott, Philadelphia, Pa.). The hemagglutinin (H) envelope glycoprotein mediates particle attachment (Dorig et al., (1993) Cell 75(2), 295-305; Erlenhoefer et., (2001) J Virol 75(10), 4499-4505; Naniche et al., (1993) J Virol 67(10), 6025-6032; Tatsuo et al., (2000) Nature 406(6798), 893-897), followed by membrane fusion orchestrated by the fusion (F) envelope protein (Lamb et al., (2006) Virology 344(1), 30-37). Viral gene expression and subsequent genome replication then take place in the cytosol (Griffin, D. E. (2001) Measles Virus, 4 Ed., Lippincott, Philadelphia, Pa.). Both processes are mediated by the viral RNA-dependent RNA polymerase (RdRp) complex, which consists minimally of a homotetramer of the viral phosphoprotein (P) and a single polymerase (L) protein (Bourhis et al., (2006) Virology 344(1), 94-110; Lamb & Kolakofsky, D. (2001) Paramyxoviridae: The viruses and their replication. In: Knipe, D. M.& Howley, P. M. (eds). Fields Virology, 4 Ed., Lippincott Williams & Wilkins, Philadelphia). Sole target for RdRp is a ribonucleoprotein complex of viral RNA encapsidated by the MV nucleocapsid (N) protein (Bourhis et al., (2006) Virology 344(1), 94-110), minimizing the presence of naked genomic RNA in the host cell. Considering that human and animal tissues lack a known homologue of the RdRp or the fusogenic envelope proteins, the polymerase complex and components of the entry machinery constitute particularly attractive targets for virus-specific small molecule inhibitors.
Despite its critical role in the viral life cycle, our mechanistic understanding of the MV RdRp is still limited and the structural characterization of its components is sparse. An abundance of structural disorder has been found in the MV N and P proteins, and no paramyxovirus polymerase has been purified thus far. In addition to their therapeutic potential, small molecule compounds targeting the MV RdRp complex may thus constitute viable tools for a better molecular and structural characterization of the viral replication machinery.
In contrast to the RdRp, considerable structural information is available for the paramyxovirus attachment and fusion protein, including structures of the latter in both the pre- and intermediate to post-fusion conformation. Relying on the molecular characterization of MV strains with distinct cytopathicity and a structural model of the MV F protein, we have in previous work identified a new class of MV fusion inhibitors, substituted anilides, in a structure-based drug design approach. The lead compound of this inhibitor class, AS-48 shows activity in the low micromolar range (IC50=0.6 to 3.0 μM) against a panel of MV field isolates. A single Sub-Saharan isolate is resistant to inhibition by AS-48, however, and in vitro adaptation has resulted in the appearance of characteristic escape mutants after four to seven passages, suggesting that resistance may emerge rapidly in the field. The identification of additional drug candidates against MV with diverse target characteristics is therefore imperative. In addition to counteracting pre-existing resistance, combined administration of compounds with different target sites may reduce the rate of viral escape or result in impaired fitness of virions which develop multiple resistance.